Prostaglandin endoperoxide H synthases-1 and -2 (PGHSs-1 and -2) catalyze the committed step in prostaglandin (PG) synthesis and are targets of nonsteroidal anti-inflammatory drugs and "COX-2 inhibitors". Each PGHS isoform subserves different physiological functions, but it is not known why it is necessary to have two isoforms. There are instances where PGHS-1 and -2 are co-expressed in cells at similar levels and the same subcellular sites and yet PGHS-2 produces PGs while PGHS-1 does not. These observations would argue against PGHS-2 being an inducible substitute or supplement for PGHS-1. More likely, the unique biological functions of PGHSs-1 and -2 result, at least in part, from structural/catalytic differences between the proteins themselves (i.e. PGHS-2 can do something that PGHS-1 cannot do, and conversely). PGHS-2 has three unique features: (a) an ability to oxygenate arachidonic acid (AA) esters efficiently; (b) the capacity to oxygenate AA at low concentrations of AA and activating peroxides; and (c) an 18 amino acid cassette near its C-terminus. There are two major goals of our proposed research. The first is to characterize further the basic structural and enzymatic properties of the cyclooxygenase and peroxidase activities of PGHSs-1 and -2. In the case of the cyclooxygenase, we will test predictions for the functions of specific active site amino acids in catalysis using a combination of mutagenic and x-ray crystallographic studies. With the peroxidase, we will use mutagenic, kinetic and computational studies to determine the basis for the unusual peroxide substrate specificity of PGHSs and for the difference between peroxide activation of the cyclooxygenase activities of PGHS-1 vs -2; in parallel, we will determine the roles of the peroxidase activities of PGHS-1 vs -2 in regulating cyclooxygenase activity in intact cells. Our second major goal is to determine if two of the distinctive properties of PGHS-2 (i.e. reactivity with AA esters or the C-terminal cassette) are essential for its novel biological actions. We will prepare and characterize the reproductive and developmental phenotypes of knock-in mice with mutations in their PGHS-2 gene such that they (a) can use AA but not esterified AA as a substrate (R120Q PGHS-2) or (b) lack the C-terminal cassette (delta581-598 PGHS-2) to determine if the knock-in mice are the same as PGHS-2 knock out mice.